Temporal regulation of neuropilin-1 expression and sensitivity to semaphorin 3A in NGF- and NT3-responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.     

Growth cones integrate signaling from multiple guidance cues.

Nerve growth factor (NGF) and semaphorin3A (Sema3A) are guidance cues found in pathways and targets of developing dorsal root ganglia (DRG) neurons. DRG growth cone motility is regulated by cytoplasmic signaling triggered by these molecules. We investigated interactions of NGF and Sema3A in modulating growth cone behaviors of axons extended from E7 chick embryo DRGs. Axons extending in collagen matrices were repelled by Sema3A released from transfected HEK293 cells. However, if an NGF-coated bead was placed adjacent to Sema3A-producing cells, axons converged at the NGF bead. Growth cones of DRGs raised in 10(-9) M NGF were more resistant to Sema3A-induced collapse than when DRGs were raised in 10(-11) M NGF. After overnight culture in 10(-11) M NGF, 1-hr treatment with 10(-9) M NGF also increased growth cone resistance to Sema3A. Pharmacological studies indicated that the activities of ROCK and PKG participate in the cytoskeletal alterations that lead to Sema3A-induced growth cone collapse, whereas PKA activity is required for NGF-mediated reduction of Sema3A-induced growth cone collapse. These results support the idea that growth cone responses to a guidance cue can be modulated by interactions involving coincident signaling by other guidance cues.     

Neurotrophin-3 promotes the survival of a limited subpopulation of cutaneous sensory neurons

In the chick embryo, exogenous neurotrophin-3 (NT3) is sufficient to promote the differentiation of proprioceptive afferents even in the absence of limb muscle targets. To determine if NT3 can promote the differentiation of this phenotype in afferents with cutaneous targets, we analyzed the effects of chronic NT3 on cutaneous and muscle sensory neurons that express trkC, a receptor for NT3. In normal embryos, retrograde labeling and immunohistochemistry showed that about 75% of large-diameter muscle afferents express trkC, whereas only about 7% of large-diameter cutaneous afferents express this protein. After chronic treatment with NT3 during the cell death period, both populations of trkC(+) neurons were increased approximately twofold. Because this treatment is known to block cell death in sensory neurons, these results indicate that NT3 can promote the survival of both proprioceptive afferents and cutaneous afferents. To examine the phenotype of the cutaneous afferents rescued by NT3, we analyzed their projections and connections using transganglionic labeling and electrophysiological recording. The results indicate that exogenous NT3 neither altered the pattern of spinal projections nor caused cutaneous afferents to form monosynaptic connections with motor neurons. These results demonstrate that selective cell death does not contribute to the modality-specific pattern of spinal innervation and suggest that proprioceptive afferents may innervate muscle selectively.     

Ephrin-A5 inhibits growth of embryonic sensory neurons

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.     

Identification and characterization of a novel mouse plexin,plexin-A4

Plexins belonging to the plexin-A subfamily form complexes with neuropilins and propagate signals of class 3 semaphorins into neurons, even though they do not directly bind the semaphorins. In this study, we identified a new member of the plexin-A subfamily in the mice, plexin-A4, and showed that it was expressed in the developing nervous system with a pattern different to that of other members of the plexin-A subfamily (plexin-A1, plexin-A2 and plexin-A3). COS-7 cells coexpressing plexin-A4 with neuropilin-1 were induced to contract by Sema3A, a member of the class 3 semaphorin. Ectopic expression of plexin-A4 in mitral cells that are originally insensitive to Sema3A resulted in the collapse of growth cones in the presence of Sema3A. These results suggest that plexin-A4 plays a role in the propagation of Sema3A activities.     

Muscle‐derived neurotrophin‐3 reduces injury‐induced proprioceptive degeneration in neonatal mice

During perinatal development, proprioceptive muscle afferents are quite sensitive to nerve injury. Here, we have used transgenic mice that overexpress neurotrophin‐3 (NT‐3) in skeletal muscle (myo/NT‐3 mice) to explore whether NT‐3 plays a neuroprotective role for perinatal muscle afferents following nerve injury. Measurements of NT‐3 mRNA using RT‐PCR revealed that levels of endogenous NT‐3 mRNA in wild‐type muscles remained constant during the first postnatal week following nerve crush or nerve section on postnatal day (PN) 1. In comparison, myo/NT‐3 mice had significantly elevated levels of NT‐3 mRNA that were maintained or increased following injury. To assess whether muscle‐derived NT‐3 could prevent injury‐induced neuronal death, neuron survival in the DRG was analyzed in mice 5 days after sciatic nerve crush on PN3. Retrograde prelabeling of muscle afferents and parvalbumin immunocytochemistry both revealed that overexpression of NT‐3 in muscle significantly reduced neuronal loss following injury. Similar neuroprotective effects of NT‐3 were observed in wild‐type mice injected with exogenous NT‐3 in the gastrocnemius muscles. To test whether NT‐3 could prevent muscle spindle degeneration, spindle number and morphology were assessed 3 weeks after sciatic nerve crush or section on PN1. No spindles were present in either wildtype or myo/NT‐3 muscles after nerve section, demonstrating that NT‐3 overexpression cannot maintain spindles following complete denervation. Moreover, NT‐3 overexpression could not prevent moderate spindle loss in muscle and did not stimulate new spindle formation following nerve crush. Our results demonstrate that in addition to its early actions on sensory neuron generation and naturally occurring cell death, NT‐3 has important neuroprotective effects on muscle afferents during postnatal development. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 198–208, 2002; DOI 10.1002/neu.10024     

Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.     

Ectopic adenoviral vector-directed expression of Sema3A in organotypic spinal cord explants inhibits growth of primary sensory afferents

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949-959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.     

Semaphorin 3d and Semaphorin 3e Direct Endothelial Motility through Distinct Molecular Signaling Pathways

Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors and since then have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) has been shown previously to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. We showed recently that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e affect human umbilical vein endothelial cells similarly but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1)-blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d-mediated, but not Sema3e-mediated, inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in human umbilical vein endothelial cells, and inhibition of the PI3K/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.     

Vocal control area‐related expression of neuropilin‐1, plexin‐A4, and the ligand semaphorin‐3A has implications for the evolution of the avian vocal system

The avian vocal system is a good model for exploring the molecular basis of neural circuit evolution related to behavioral diversity. Previously, we conducted a comparative gene expression analysis among two different families of vocal learner, the Bengalese finch (Lonchura striata var. domestica), a songbird, and the budgerigar (Melopsittacus undulatus), a parrot; and a non‐learner, the quail (Coturnix coturnix), to identify various axon guidance molecules such as cadherin and neuropilin‐1 as vocal control area‐related genes. Here, we continue with this study and examine the expression of neuropilin and related genes in these species in more detail. We found that neuropilin‐1 and its coreceptor, plexin‐A4, were expressed in several vocal control areas in both Bengalese finch and budgerigar brains. In addition, semaphorin‐3A, the ligand of neuropilin‐1, expression was not detected in vocal control areas in both species. Furthermore, there was some similar gene expression in the quail brain. These results suggest the possibility that a change in the expression of a combination of semaphorin/neuropilin/plexin was involved in the acquisition of vocal learning ability during evolution.     

Deletion of Sema3a or plexinA1/plexinA3 Causes Defects in Sensory Afferent Projections of Statoacoustic Ganglion Neurons

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.     

Ventral migration of early-born neurons requires Dcc and is essential for the projections of primary afferents in the spinal cord

Neuronal migration and lamina-specific primary afferent projections are crucial for establishing spinal cord circuits, but the underlying mechanisms are poorly understood. Here, we report that in mice lacking Dcc (deleted in colorectal cancer), some early-born neurons could not migrate ventrally in the spinal cord. Conversely, forced expression of Dcc caused ventral migration and prevented dorsolateral migration of late-born spinal neurons. In the superficial layer of the spinal cord of Dcc-/- mutants, mislocalized neurons are followed by proprioceptive afferents, while their presence repels nociceptive afferents through Sema3a. Thus, our study has shown that Dcc is a key molecule required for ventral migration of early-born neurons, and that appropriate neuronal migration is a prerequisite for, and coupled to, normal projections of primary afferents in the developing spinal cord.     

Attractive and permissive activities of semaphorin 5A toward dorsal root ganglion axons in higher vertebrate embryos

Elongation of the efferent fibers of dorsal root ganglion (DRG) neurons toward their peripheral targets occurs during development. Attractive or permissive systems may be involved in this elongation. However, the molecular mechanisms that control it are largely unknown. Here we show that class 5 semaphorin Sema5A had attractive/permissive effects on DRG axons. In mouse embryos, Sema5A was expressed in and around the path of DRG efferent fibers, and cell aggregates secreting Sema5A attracted DRG axons in vitro. We also found that ectopic Sema5A expression in the spinal cord attracted DRG axons. Together, these findings suggest that Sema5A functions as an attractant to elongate DRG fibers and contributes to the formation of the early sensory network.     

Attractive and permissive activities of semaphorin 5A toward dorsal root ganglion axons in higher vertebrate embryos

Elongation of the efferent fibers of dorsal root ganglion (DRG) neurons toward their peripheral targets occurs during development. Attractive or permissive systems may be involved in this elongation. However, the molecular mechanisms that control it are largely unknown. Here we show that class 5 semaphorin Sema5A had attractive/permissive effects on DRG axons. In mouse embryos, Sema5A was expressed in and around the path of DRG efferent fibers, and cell aggregates secreting Sema5A attracted DRG axons in vitro. We also found that ectopic Sema5A expression in the spinal cord attracted DRG axons. Together, these findings suggest that Sema5A functions as an attractant to elongate DRG fibers and contributes to the formation of the early sensory network.     

SEMA3A regulates developing sensory projections in the chicken spinal cord

The present study explores the role of SEMA3A (collapsin-1) in the temporal and spatial regulation of developing sensory projections in the chick spinal cord. During development, SEMA3A mRNA (SEMA3A) is first expressed throughout the spinal gray matter, but disappears from the dorsal region when small caliber (trkA(+)) sensory axon collaterals first grow into the dorsal horn. In explant cultures of spinal cord segments with attached sensory ganglia, the spatial extent of SEMA3A expression varied in different explants, but in each case the growth of trkA(+) sensory collaterals was largely excluded from areas of SEMA3A expression. To test if SEMA3A had a direct effect on sensory axon growth, we injected recombinant protein into the explants before placing them in culture. Increased levels of SEMA3A substantially reduced the ingrowth of trkA(+) axons, whereas trkC(+) axon collaterals were not affected. Consistent with the insensitivity of trkC(+) collaterals to SEMA3A, these collaterals did not express neuropilin-1, a receptor for SEMA3A. The inhibitory effects of SEMA3A on trkA(+) axons within the spinal cord suggests that the fall in SEMA3A expression in the dorsal horn may contribute to the initiation of growth of these axons into gray matter. In addition, the observation that trkA(+) axons frequently grew close to but rarely over areas of SEMA3A expression suggests that semaphorin may act principally as a short-range guidance cue within the spinal cord.